Burden and typing of rotavirus group a in children with acute gastroenteritis in shiraz, southern iran.

Background Human Rotavirus is a significant cause of severe gastroenteritis in infants and young children worldwide. In recent years, Rotavirus genotyping by RT-PCR has provided valuable information about the diversity of Rotaviruses circulating worldwide. The purpose of the present study is to monitor the prevalence of the different G types of Rotaviruses circulating in Shiraz, Southern Iran and detect any uncommon or novel types. Methods During the period from December 2007 to November 2008, a total of 138 stool samples were collected from children less than 5 years old who were hospitalized for acute gastroenteritis. Rotavirus-associated diarrhea was investigated in fecal specimens with enzyme immunoassays (EIA). Rotavirus-positive specimens were typed by the Nested RT-PCR and by using different types of specific primers. Results Out of the 138 collected samples, 34.78% (48 cases) tested positive for Rotavirus. The frequency of G1, G2 and G4 types was 6.25%, 2.08% and 27.08%, respectively. Mixed and non-typeable infections were detected in 33.34% and 31.25% of hospitalized children with acute diarrhea, respectively. This is the first time mixed Rotavirus infections with G1/G3 have been reported in Iran. Conclusion The high frequency of Rotavirus detection indicates the severity and the burden of Rotavirus disease may be able to reduce through the implementation of an effective vaccine and continual surveillance for the detection of Rotavirus genotypes circulating in other regions of Iran. Regarding to the noticeable frequency of non-typeable and mixed infections, it is suggested to use the other specific primers and further studies to detection of other novel and unusual types.


Introduction 1Human
Rotavirus is the leading cause of severe gastroenteritis in infants and young old. 2 Nearly every child will infect with Rotavirus disease by age 5 years, one in five will require a clinic, one in 50 will be hospitalized, and one in 205 will die from this disease. 3

Specimen collection
In this study a verbal consent was taken from either parent of the enrolled child prior to the interview and collection of stool samples. From December 2007 to November 2008, a total of 138 stool specimens were collected during the course of treatment from children under 5 years of age who were hospitalized with acute gastroenteritis in Shahid Dastgheib and Nemazee hospitals in Shiraz, Southern Iran. All the fecal specimens were transported to the infectious disease unit laboratory and stored at -70°C until the time of assay. All samples underwent only one cycle of thawing and freezing prior to characterization. A standard structured questionnaire was used to obtain the information regarding the age, sex, duration of hospital stay, severity of clinical symptoms and type of feeding (as breast/bottle feeding) for each case.

Rotavirus detection
All samples were screened for group A Rotavirus by enzyme immunoassay (EIA) (Rotavirus Ag ELISA, DRG, Germany), according to the manufacturer's instructions.

Viral RNA extraction
Genomic RNA was extracted with a commercially available mixture of phenol and guanidine thiocyanate (RNX-Plus kit, CinnaGen, Tehran, Iran). Isolation of whole RNA was performed according to the manufacture s protocol. Briefly, 500 µl RNX-Plus solution was mixed with a 20% stool suspension in phosphate buffer saline (PBS) at a pH of 7.2 at a volume ratio of 3 to 1. After complete dissociation of nucleoprotein complexes, chloroform was added to the mixture followed by vigorous shaking and centrifugation at 12,000 g for 15 min at 4°C. The upper aqueous phase was transferred to a fresh tube and the RNA precipitated by mixing it with isopropanol. The supernatant was removed and the RNA pellet was washed once with 75% ethanol. The RNA pellet was then briefly air dried and dissolved in diethyl pyrocarbonate (DEPC) treated water.

Reverse transcription-polymerase chain reaction
Briefly, 5 µl of dsRNA was added to a mix of DMSO, 5X RT buffer, dNTPs, primers Beg9, End9, 25 and DW, denatured at 97°C for 5 min. Then RT enzyme and RNase inhibitor were added to make a final volume of 20 µl. The RT-PCR reaction was carried out for 60 min at 42°C to produce the complementary (cDNA) used for PCR amplification Rotavirus.

Nested multiplex PCR for G genotyping
The G-typing was performed according to Rotavirus detection and typing protocol provided by WHO. 25 Briefly, the first round VP7 consensus PCR was carried out with 10 l of cDNA in 40 l of the VP7 reagent mixtures. The cycling parameters used were: 30 cycles at 94°C for 1 min, 42°C for 2 min, 72°C for 2 min, and a final extension at 72°C for 5 min. The second round VP7 multiplex PCR was carried out with 5 l of first round VP7 amplicons in 40 l of the second round VP7 reagent mixtures. Cycling was done with 20 cycles of the same cycling profile as the first reaction. The PCR mixtures contained 10x PCR buffer, MgCl2 (50 mM), deoxynucleoside triphosphates (10 mM), primers (10 pmol), and Taq DNA polymerase (1U). The amplified product was visualized by gel electrophoresis using 2% agarose gel containing ethidium bromide (10 g/mL). The 100 bp DNA ladder (GeneRuler , Fermentas life science) was used as a molecular weight standard. Primer sequences are shown in Table   1.

Data analysis
Data was statistically analyzed by SPSS version 15 (SPSS Inc., Chicago, IL, USA). Statistical analysis 2 test was used to analyze the data obtained to the age group, sex, G types and seasonal distribution of the group A Rotavirus and also type of feeding. Fisher s exact test was used to analyze the clinical symptoms. P value <0.05 was considered statistically significant.

Rotavirus detection
A total of 48 (34.78%) diarrheic fecal specimens were confirmed as Rotavirus positive using EIA assay.

Discussion
Rotavirus is a significant cause of diarrhea in developed and under developed countries in children under five years of age, and it is essential to determine the circulating genotypes and their temporal and geographical variations.  34 and Iran (60%), 35 and higher than studies conducted in Ireland, 36 Indonesia, 37 Africa, 38 India, 39 with the prevalence of 28.5%, 23%, 21.4% and 21%, respectively. In the current survey, we documented the first case of G1/G3 mixed infection in Iran. This G type has been reported in young children with gastroenteritis in Mexico. 40  However the G1 type was observed only in 6.25% of all children with Rotavirus diarrhea. We detected the G2 type in the specimens of only 2.08% of patients with acute diarrhea, which is in contrast with studies conducted in Italy, Sierra Leone and India, which demonstrated that the G2 type is one of the most prevalent Rotavirus types. 10,19,20,44 In the current study, neither the G3 nor the G8 types were detected individually. These findings are distinct from those results observed in Sierra Leone, 10 Turkey, 13 Malawi, 21 and China, 45 where these G types have been identified as the most common types in children. In recent years there has been an increase in research of the importance of G9 type in many countries including Latin America, 12 Iran, 16 Albania, 19 Brazil, 37 Cuba, 46 and Tanzania. 47 However, the G9 type was not detected during the study. Investigations in developing and industrialized countries have demonstrated the need for new generations of Rotavirus vaccines to include G9 strains due to the increasing emergence of this type of group A Rotavirus. In conclusion this study provides information on the epidemiology and description of the Rotavirus G types circulating among Iranian children with acute diarrhea. Our results indicate that gastroenteritis caused by Rotavirus in the country is a significant health problem, particularly among children less than 2 years of age and during the cold season. These data will be useful for making an informed decision about the introduction of Rotavirus vaccine in Iran and provides a baseline data for future vaccine studies.

Financial support
None declared.

Conflict of interest
None declared.

Acknowledgment
Our special thanks and appreciation go to the Islamic Azad University, Jahrom Branch, who provided executive support of this Project. We also thank the thoughtful of Mehdi Kargar and Dr. Ramin Yaghobi who assisted us in performing this project.

Author Contribution
KM carried out the design of the study, coordination and scientific consultation at Islamic Azad University of Jahrom. JT and NA participated in sampling and practical methods. All authors read and approved the final manuscript.